Imaging has played an important part in the analysis of specimen, and in particular, in the analysis of biological specimen. The tools most commonly used for imaging biological molecular architectures are immunofluorescence microscopy (IFM), immunoelectron microscopy (IEM) and genetically encoded fluorescent protein markers and reporters (XFPs). These powerful and widely used tools have led to discoveries that point to the dependence of cell and tissue function upon the very finest details of molecular architecture, and further that demonstrate the extremely variable and dynamic molecular architectures of specimen.
IFM, IEM and XFP imaging methods present challenges, however, and many important aspects of cell and tissue molecular architecture are difficult to explore using previous approaches. For instance, while XFPs have provided unique opportunities for imaging live cell dynamics, they require the expression of transgenes and are thus impractical for the study of human clinical specimens. XFPs also entail substantial probability of perturbing the molecular architectures of interest, and offer generally limited molecular multiplexing capabilities.
While both IFM and IEM are generally confined to use on fixed cells and tissues, these techniques do not require transgene expression, and are thus readily applied to human cell and tissues. IFM provides an ease of use and substantial multiplexing capacity that have made it one of the most widely used tools of cell biology research. However, IFM has generally been limited in resolution and quantitative interpretability.
IEM provides a relatively high resolution and has been useful in its power to discern very fine details of molecular architecture. However, IEM has proven difficult to apply to three-dimensional architectures, has limited multiplexing capacity, and has had very limited volumetric imaging capabilities, particularly as applied to tissue-scale problems.
Generally, imaging specimen, and particular, imaging biological specimen has been challenging.